Regeneration medicine and tissue engineering are typically proposed as an ideal solution to overcome limitations due to lack of tissues and/or organs for graft substitutes. The tissue engineering is a technique in which specific cells separated and cultured from a patient are adhered to an implant made of biocompatible/biodegradable material, and the implant is organized through biochemical stimulation using a bioactive factor or physical stimulation using a bioreactor.
In other words, the artificial organ engineered and manufactured according to the above method is similar to bio-tissues of a human body, thus to have a high potential ability as a graft substitute for autologous tissue implant. As described above, the tissue engineering may be defined with three elements including a cell, stimulant and implant. The present invention has focused on the implant, in particular, an ultra-thin silk composite implant manufactured by using biomaterials, and investigated characteristics thereof.
Since the silk has been used as a sawing yarn for several centuries, it is possible to have a role of favorable bio-materials in the tissue engineering applications. Korean Patent Laid-Open Publication No. 2010-0121169 relates to an artificial eardrum fabricated using silk protein and a manufacturing method thereof, and proposes that an implant obtained in the manufacturing process with addition of any additive may have a thickness of 80 to 120 μm thus to apply the same to a tissue that requires a thin film such as an eardrum. However, the above technique could not be used in a tissue that requires an ultra-thin film of 20 μm or less such as a cornea, hence having a limitation in applications.
Korean Patent Laid-Open Publication No. 2011-0104584 relates to a composite support including silk and collagen and a method for manufacturing the same. In this case, the above support may form a structure including a woven silk tube layer and a collagen layer inside the tube, thus to provide a three-dimensional support useable as a matrix for regeneration of ligaments and tendons, reconstruction of damaged muscles, or the like. However, the above technique involves limitations in applying the above support to ultra-thin films such as cornea, retina, eardrum, etc. and some applications requiring transparency.
Meanwhile, Bombyx mori (B. mori)-derived silk consists of two different proteins, that is, fibroin and sericin.
Herein, the fibroin is a protein constituting about 75% in silk cocoon and includes at most 90% of insoluble proteins such as glycine, alanine and serine. Further, the fibroin does not occur an immune reaction during in vivo transplant, may control a degree of degradation, and has excellent mechanical strength and good transparency to oxygen and water.
Further, silk fibroin supports adhesion and growth of human limbal stem cells and fibroblast cells, and is used in a wide range of applications such as a wound dressing agent, enzyme immobilization membrane, cell medium, artificial skin, soft contact lens, etc. Furthermore, many researchers have reported that a two-dimensional silk fibroin film could provide desired biological synthesis performance.
In addition, sericin constitutes the remaining 25% in the silk cocoon, but causes an occurrence of the immune reaction and allergic response, thus being typically used after a refining process of removing the sericin. However, sericin is a hydrophilic protein and has a strong polar chain, and further is a biodegradable material to provide a variety of advantages such as anticoagulation, antioxidation, and anti-wrinkling properties. Further, the researchers have recently reported that sericin can prevent cell apoptosis, inhibit cancer generation, reinforce adhesion and proliferation of fibroblast cells, and be applicable as a component of the cell medium.
Meanwhile, the above-described silk fibroin may be used in the tissue engineering by dissolving the refined silk fibroin in an organic solvent after lyophilizing, then, preparing the same into a film through a casting process. The prepared silk fibroin film may possibly contain the organic solvent residue remained therein, and the organic solvent residue toxically may act on the cell and the body in vivo during transplant thus to affect the adhesion and growth of the cells. Accordingly, it has been raised a requirement for an ultra-thin film composite implant for implanting, wherein the implant has no cytotoxicity, minimizes an effect on the cell growth, does not cause toxicity in vivo during transplant, and can be implanted in any case where needs the ultra-thin film such as corneal endothelial transplantation.
The above subject matters described as a background of art have been proposed to more concretely understand the background of the present invention only, however, it should not be construed or recognized to correspond to the prior art known to those skilled in the art to which the present invention pertains.